Otitis media (OM) is the most common reason that an American child receives antibiotics or undergoes a general anesthetic. OM is the leading cause of pediatric hearing loss and can lead to language, speech and behavioral problems. OM disproportionately affects children of color and hinders the full participation of women in the workforce. In this continuation application, we propose to extend our paradigm-shifting discovery that chronic OM is actually a biofilm illness. The Center for Genomic Sciences and the Center for Biofilm Engineering have a 6-year history of collaborative effort, and have published the results in Science, JAMA, and Scientific American. In the first phase of these investigations we developed expertise with both novel biological reagents and the construction and operationalization of extensive new facilities and instrumentation platforms. The results of these led to the development of a new overarching theoretical construct, termed Bacterial Plurality, which states that chronic bacterial pathogenesis and persistence reside at the population level rather than within individual bacterium. In this continuation, we will be applying cutting edge technology focused by a new theoretical understanding of chronic infections to provide a global framework for understanding persistent Pseudomonas aeruginosa (PA)-induced chronic OM and otorrhea. The goals of the proposed work are: 1) to study the genetic interplay between the host mucosa and a mucosal biofilm in our PA-induced chronic OM chinchilla model; 2) test the theory of bacterial plurality using an evolutionary fitness model. Using the multi-platform, massively parallel, integrated systems developed during the first grant cycle, we will quantitate the degree of genomic plasticity by direct genome sequencing of PA, and divine the extent of variation in gene expression among the many bacterial envirovars that these organisms can adopt. A discovery that has immediate clinical relevancy is that biofilm antibiotic resistance is mediated largely by bacterial metabolic resistance owing to limiting nutrient conditions within the core of the biofilms. [unreadable] [unreadable]